Transformation of plant cells by an Agrobacterium-mediated method involves exposing plant cells and tissues to a suspension of Agrobacterium cells that contain certain DNA plasmids. These plasmids have often been specifically constructed to contain transgenes that will express in plant cells (see, for example, U.S. Pat. No. 5,034,322). Most often, one or more of the transgenes is a selectable marker transgene that permits plant cells to grow in the presence of a positive selection compound, such as an antibiotic or herbicide. These cells can be further manipulated to regenerate into whole fertile plants.
Methods for introducing transgenes into plants by an Agrobacterium-mediated transformation method generally involve a T-DNA (transfer DNA) that incorporates the genetic elements of at least one transgene and transfers those genetic elements into the genome of a plant. The transgene(s) are typically constructed in a DNA plasmid vector and are usually flanked by an Agrobacterium Ti plasmid right border DNA region (RB) and a left border DNA region (LB). During the process of Agrobacterium-mediated transformation, the DNA plasmid is nicked by an endonuclease, VirD2, at the right and left border regions. A single strand of DNA from between the nicks, called the T-strand, is transferred from the Agrobacterium cell to the plant cell. The sequence corresponding to the T-DNA region is inserted into the plant genome.
Integration of the T-DNA into the plant genome generally begins at the RB and continues to the end of the T-DNA, at the LB. However, endonucleases sometimes do not nick equally at both borders. When this happens, the T-DNA that is inserted into the plant genome often contains some or all of the plasmid vector DNA. This phenomenon is referred to as “read-through.” A desired approach is often that only the transgene(s) located between the right and left border regions (the T-DNA) is transferred into the plant genome without any of the adjacent plasmid vector DNA (the vector backbone). Vector backbone DNA contains various plasmid maintenance elements, including for example, origin of replications, bacterial selectable marker genes, and other DNA fragments that are not required to express the desired trait(s) in plants.
Chen et al. (Functional Plant Biology (2005) 32:671-681) assert that they have developed a T-DNA method designed to reduce the frequency of transformed plants with multiple copies of the T-DNA. Chen et al. does not provide a 2T-DNA system and was not designed to specifically eliminate the frequency of transformed plants with linked insertions. Moreover, Chen et al. does not provide a strategy to eliminate vector backbone correlated with linked insertions. The approach of Chen et al. does not permit the transcriptional unit to be oriented in any direction and was not designed to produce single copy plants that are marker-free.